This work was funded by the American Diabetes Association (ADA), Grant JF1-10-14 (MDiF). We would like to thank the personnel of the Laboratory of Animal Resources at WSU. Authors gratefully acknowledge assistance with the statistical analysis of data from Neil Paton, Ph.D.

TNTs of mice and rats are determined by using the incremental hot-plate analgesia meter [iHPAM, IITC Inc. Life Science (Woodland Hills, CA)]. The equipment consists of several components: an aluminum plate (10 x 20 cm) with the heating system underneath and a Plexiglas observation chamber above; a heat controlling unit, software for data acquisition (IITC Part # Soft Series 8), a PC (personal computer) and a footswitch for remote start, stop or reset control of the unit. The heating system also allows starting/stopping/resetting of the heating process manually through a keypad in front of the equipment. The iHPAM can be set to various heating rates, standby/cutoff temperatures.
1. Equipment Set-up
<ol>
	<li>Before testing animals, visually inspect the unit to corroborate the chosen heating rates (e.g., 6 &deg;C/min), initial standby and final cut-off temperatures (e.g., 28 &deg;C and 55 &deg;C, respectively).</li>
	<li>Verify all connections. To keep electronic copy of the results, the unit must be connected to a computer running Software Series 8. Alternatively, hard copies of the results can be printed out via the serial port of unit.</li>
	<li>Test the equipment. Press the footswitch once to make sure the Software Series 8 records the data: initial standby, final cutoff temperatures and time in seconds required to reach the cut-off temperature.</li>
	<li>As a further control test, the investigators involved in the experiments may need to experience the device on themselves. Withdrawing the hand from the metal plate at the moment of pain perception (46-48 &deg;C) must neither cause harm to the skin nor produce any later discomfort.</li>
	<li>Once the unit has been set and the results from steps 1.3 and 1.4 recorded they can be compared. This is helpful to detect potential problems that could invalidate the use of the unit. For instance, discrepancies between initial standby/final cut-off temperatures and the actual reading of the unit, and/or differences between temperatures displayed by the unit and the actual plate temperature required for hand withdrawal.</li>
</ol>
2. Testing Normal Animals
Either young or adult Wistar rats [post-natal (p) age 21-25 days], or mice (p21-25) of any genetic background or gender can be used. The mice used in our experiments are of the strain C57BL/6J.
<ol>
	<li>The night before the experiments to be performed, acclimate the animals by placing their cages on the bench where the iHPAM is placed. Two hours before performing the tests, turn on the iHPAM to familiarize the animals with the background noise of the unit. At this point, the iHPAM can be set up and tested to verify proper function (see Steps 1). Food and water is provided ad libitum throughout the test, except when the animal is in the observation chamber (see Step 2.1). 
	Note: It is important not to assume that the laboratory conditions in behavioral experiments are controlled by default. Special attention should be paid to genetic and environmental factors that may influence the outcome of the behavioral studies (reviewed in 7).</li>
	<li>Gently place a normal mouse/rat in the Plexiglass observation chamber on top of the heating plate of the unit. Allow the animal to acclimate to the warmed surface (set at 28 &deg;C) until the animal shows a comfortable behavior. 
	Note: Under this conditions normal adult rats show an initial exploratory behavior, occasionally standing up towards the lid of the observation chamber while sniffing. After a few minutes, rats may show casual bouts of grooming and a relaxed behavior. Sometimes, rats lay with a relaxed body posture (prone extension) while casually sniffing the surroundings. Normal mice are usually more active than rats. However, mice show a relaxed exploratory behavior after few minutes in the observation chamber. It is exceptionally rare to observe either escaping behaviors (e.g., jumping, trying to escape from the observation chamber) or defensive attitudes (e.g., squealing). It is also important to note that mice can communicate pain from one mouse to another8. Therefore, it may be important to test mice placed in separate cages.</li>
	<li>Place mirrors in the back of the observation chamber, to permit observations at all angles. A tripod holding a digital video camera may be of help to determine the exact moment at which nocifensive (pain avoiding) behavior had occurred.</li>
	<li>Once the animal is comfortable in the observation chamber heat up the plate by pressing the footswitch. When the plate temperature heats up at a rate of 6 &deg;C/minute, normal animals exhibit nocifensive behavior involving either hind-paw* usually temperatures raging from 46 to 48 &deg;C3,5,9,10. Thus, the animal is typical removed form the observation chamber after approximately 3 minutes. 
	Note: Observing the typical nocifensive response of a rat or mouse i.e., hind-paw licking, while shaking and/or lifting, is enough to terminate the thermal stimulus. This is achieved by pressing the footswitch. At this moment, the software records the plate's temperature and the plate immediately cools down until standby temperature (28 &deg;C) is reached (~0.5 min). This temperature i.e., the one that evokes any nocifensive reaction is regarded as the noxious heat threshold (TNT) of the normal (control) animal. 
	*Sometimes, particularly in mice, nocifensive behavior to noxious temperature may first be observed in one or both forepaws (e.g., licking while sitting), few seconds before hind-paws. However, fore-paw licking is a normal component of the grooming behavior. Hence, only hind-paw reactions are assessed. In occasions mice may groom their hind-paws as well. However, normal grooming do not last more than a few seconds, does not involve intensive licking and usually ends after cleaning its claws.</li>
	<li>Gently remove the animal from the plate and place it in its respective cage. The heat threshold measurement is then repeated for other control (normal) animal. 
	Note: TNTs can be determined in a single animal at intervals of 1-2 minutes without removing the animal from the observation chamber. TNT determinations in rats or mice can be repeated several times during several days. This is particularly useful to determine the mean TNT of an individual animal as well as its variation with age.</li>
	<li>The TNT of control animals is expressed as the mean of three or more thresholds &plusmn; SEM.</li>
	<li>Statistical analysis of data obtained is performed to compare different treatment groups of mice or rats. Parametric procedures including Analysis of Variance may be appropriately used where data are normally distributed and where treatment groups are homoskedastic; when these conditions have been satisfied the following approaches can be applied. Analysis of Variance followed by the Newman-Keuls test to detect differences in multiple measurements i.e., repeated individual TNTs with/without treatment or at different time points (e.g., data in Figure 3). The paired Student's t-test may be used to compare two data groups i.e., TNTs before vs. after drug treatment; the unpaired t-test may be used to determine differences in TNT changes in drug-treated vs. vehicle-treated animals (e.g., data in Figure 4).</li>
</ol>
3. Testing Diabetic Animals
The streptozotocin (STZ)-induced diabetic rat is the most commonly used animal model to study mechanisms of PDN and to assess analgesic drugs and therapies11,12. Rats made diabetic by a single intraperitoneal dose of STZ (60 mg/kg) can be tested for thermal hyperalgesia during the 3rd week after onset of diabetes11. It is important to note that not all diabetic rats develop PDN. However, animals that do develop hyperalgesia (~50%) can be easily identified. Indeed, their TNTs are lower than normal*.
*To determine TNTs of STZ-diabetic rats, a starting plate temperature of 15 &deg;C instead of 28 &deg;C is used. The reason for these change is that most diabetic animals with PDN are expected to be hyperalgesic2,11. Therefore, they will have lower than normal noxious heat thresholds (e.g., &lt;45 &deg;C).
<ol>
	<li>A diabetic rat is placed in the observation chamber on top of the testing apparatus. The animal is allowed to acclimate to the plate surface until the animal shows a comfortable behavior. 
	Note: Typically, a diabetic rat does not show much of the normal behavior. Diabetic rats are usually hypoactive, lethargic; often showing restricted movements and slow grooming. It is rare to observe standings towards the lid of the observation chamber. After few minutes, diabetic rats may calmly stay in a corner of the chamber with casual sniffing of the surroundings.</li>
	<li>Once the diabetic animal is comfortable in the observation chamber, the plate is heated up as in Step 2.3 the diabetic rat may exhibit nocifensive behavior involving either hind/fore-paw. This usually happens at non-noxious plate temperatures (e.g., &lt;45 &deg;C), or after no more than 5 minutes in the chamber with the 6 &deg;C/min temperature increase. 
	Note: The nocifensive response of a young adult diabetic rat does not differ from that of a normal rat. However, the lowest temperature evoking such behavior is significantly lowered.</li>
	<li>See Step 2.4</li>
	<li>The mean of more than three thresholds in the day of the experiment expressed as &deg;C &plusmn; SEM is considered the noxious heat threshold of the diabetic animal. 
	Note: A similar paradigm to the one explained for normal or diabetic rats can be followed to determine the thermal anti-nocifensive effect of analgesics on heat thresholds.</li>
</ol>
4. Testing Analgesia
The anti-nociceptive properties of drugs (e.g., analgesics) as well as the determination of pharmacologic parameters such as the minimum effective dose can be easily obtained by using the steps explained above. It is predicted that proper doses of an analgesic administered to rats/mice would significantly increase their TNTs. This is particularly sensitive in animals with thermal hyperalgesia3. However, to avoid bias in the interpretation of the results, the investigators involved in the behavioral observation of an animal group must not know or be aware if the animals had been treated or not. Further, drugs to be injected by the observer may be prepared by another investigator and labeled A, B, C, etc. Dosage is provided in a printout, listing individual doses per identified animal.
Depending on the drug being tested, different doses of a potential analgesic may be administered to normal or diabetic animals 5-15 min prior behavioral tests. Proper controls must be included (i.e., age-matched animals injected with vehicle alone and/or diabetic rats without PDN) and tested using identical conditions (e.g., same starting temperature of the plate). Chronic effects of drugs can be tested as well.
5. Representative Results
The lowest plate temperature evoking nocifensive reactions in either hind-paw of young adult normal non-treated rats or mice were determined following steps 2-1 to 2-6. As shown in Figure 1, TNT of normal young adult rats and mice were 47.2 &plusmn; 0.2 &deg;C and 47.5 &plusmn; 0.5 &deg;C, respectively.
We have observed neither significant daily variation of individual's TNTs (Figure 2A) nor significant changes of TNTs among individuals of the same age (Figure 2B). TNT differences due to gender were not observed (not shown).
TNTs of young adult STZ-diabetic rats were determined in a similar fashion as for normal rats (Steps 3.1 to 3.3). However, the starting plate's temperature was set to 15 &deg;C instead of 28 &deg;C. The nocifensive behavior of STZ-diabetic rats was assessed at least three times in an individual and in a daily fashion starting eleven days post-STZ injection. As shown in Figure 3, a significant drop (p&lt;0.01) in the TNT of STZ-diabetic rats becomes evident two weeks post-STZ injection. The mean TNT of STZ-diabetic rats was 45.6 &plusmn; 0.1 &deg;C (n=16, pooled values from days 14-23).
Diabetic rats exhibiting significantly lower TNT than normal were considered to have PDN. These rats were used to test the acute anti-nociceptive properties of an analgesic (compound A). TNTs were determined in normal and diabetic rats injected with either a single dose of the compound or vehicle alone five minutes before the test. As shown in Figure 4, a single intra-peritoneal dose of the compound significantly increased TNTs of diabetic rats when compared to rats treated with vehicle alone (45.6 &deg;C vs 47.6 &deg;C, respectively). The anti-nocifensive effect of compound A was also observed in normal rats; although significant (p&lt;0.01), it was less pronounced (47.4 &deg;C vs 48.0 &deg;C, respectively).
<img alt="Figure 1" src="/files/ftp_upload/3785/3785fig1.jpg" /> 
Figure 1. Determination of TNTs of normal young adult rats or mice: The TNTs of young adult rats (p21-24) or mice (p20-21) were tested in the iHPAM following the steps outlined above. A total of 12 rats and 25 mice were tested. Measurements were performed during several days and the results were pooled. Results are expressed as mean &plusmn; SEM. The minimum and maximum TNT values recorded in these rats were 46.2 &deg;C and 48.6 &deg;C respectively. In the case of mice, the maximum and minimum TNT values recorded were 48.8 &deg;C and 44.9 &deg;C, respectively.
<img alt="Figure 2" src="/files/ftp_upload/3785/3785fig2.jpg" /> 
Figure 2. Age-dependency of TNTs in rats and mice: A) Plotted are the TNTs of four normal rats (n=4) of 21 days of age determined at least three times daily during 5 days. B) TNTs of mice littermates determined once at indicated ages. The numbers on top of each determination point represents the number of littermates used.
<img alt="Figure 3" src="/files/ftp_upload/3785/3785fig3.jpg" /> 
Figure 3. Diabetic rats become hyperalgesic 3 weeks after the onset of diabetes: TNTs of six (n=6) age matched young adult STZ-diabetic rats were determined every day starting 11 days post-induction of diabetes. Shown are daily TNT averages (mean &plusmn; SEM). Diabetic TNTs determined between 11-13 days post-STZ injection were within the normal range (red line). Two weeks after STZ injection TNTs decreased significantly to 45.1 &plusmn; 0.4 &deg;C, (p&lt;0.05).
<img alt="Figure 4" src="/files/ftp_upload/3785/3785fig4.jpg" /> 
Figure 4. Acute anti-nocifensive effects of compound B in STZ-diabetic rats. Young adult normal rats (blue group n=12) and age matched STZ-diabetic hyperalgesic rats (green group n=6) were tested in the iHPAM after intraperitoneal injection of vehicle (dark bars) or compound A (light bars). The mean TNT &plusmn; SEM for each treatment (vehicle control or compound A-treated) are shown. Asterisks denote statistical differences (p&lt;0.05).

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	<li>Baron, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+neuropathic+pain:+From+mechanisms+to+symptoms.">Peripheral neuropathic pain: From mechanisms to symptoms.</a> Clin. J. Pain. 16, S12-S20 (2000).</li><li>Calcutt, N. A., Jorge, M. C., Yaksh, T. L., Chaplan, S. 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I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+hot+plate+tests+to+quantify+antinociceptive+and+narcotic+antagonist+activities.">New hot plate tests to quantify antinociceptive and narcotic antagonist activities.</a> Eur. J. Pharmacol. 27, 1-4 (1974).</li><li>Almasi, R., Petho, G., Bolcskei, K., Szolcsanyi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+resiniferatoxin+on+the+noxious+heat+threshold+temperature+in+the+rat:+A+novel+heat+allodynia+model+sensitive+to+analgesics.">Effect of resiniferatoxin on the noxious heat threshold temperature in the rat: A novel heat allodynia model sensitive to analgesics.</a> Br. J. 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We have decided not to disclose the identity of the analgesic used in these experiments. Part of the results shown here has been presented in the 71st Scientific Sessions of the American Diabetes Association (San Diego CA) and the 47th Annual Meeting of the European Association for the Study of Diabetes (Lisbon, Portugal).

Similarly to the classic hot plate test to quantify thermal hyperalgesia4,13, the assay of nociception described here permits a fast and reliable way to quantify nocifensive behavior in rats and mice. However, contrary to classic test, the incremental hot-plate method is non-invasive and virtually stress-free. Although some restrain is necessary to perform the test (i.e., the animal must be in the observation chamber), rats or mice are accustomed to similar areas (e.g., housing cages).
<...

<table border="1">
	<tbody>
 <tr>
 <td>Name</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>Incremental Hot-Plate Analgesia Meter </td>
 <td>IITC Inc. Life Science </td>
 <td>Part #PE34 </td>
 </tr>
 <tr>
 <td>Soft Series 8</td>
 <td>IITC Inc. Life Science</td>
 <td>Part # Series8</td>
 </tr>
 <tr>
 <td>Streptozotocin</td>
 <td>Calbiochem</td>
 <td>572201</td>
 </tr>
 </tbody>
 </table>

rapid determination of the thermal nociceptive threshold in diabetic rats

Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e., increased sensitivity to noxious stimulus, and allodynia i.e., hypersensitivity to normally innocuous stimuli1. Hyperalgesia and allodynia have been studied in many different rodent models of diabetes mellitus2. However, as stated by B&ouml;lcskei et al, determination of "pain" in animal models is challenging due to its subjective nature3. Moreover, the traditional methods used to determine behavioral responses to noxious thermal stimuli usually lack reproducibility and pharmacological sensitivity3. For instance, by using the hot-plate method of Ankier4, flinch, withdrawal and/or licking of either hind- and/or fore-paws is quantified as reflex latencies at constant high thermal stimuli (52-55 &deg;C). However, animals that are hyperalgesic to thermal stimulus do not reproducibly show differences in reflex latencies using those supra-threshold temperatures3,5. As the recently described method of B&ouml;lcskei et al.6, the procedures described here allows for the rapid, sensitive and reproducible determination of thermal nociceptive thresholds (TNTs) in mice and rats. The method uses slowly increasing thermal stimulus applied mostly to the skin of mouse/rat plantar surface. The method is particularly sensitive to study anti-nociception during hyperalgesic states such as PDN. The procedures described bellow are based on the ones published in detail by Alm&aacute;si et al 5 and B&ouml;lcskei et al 3. The procedures described here have been approved the Laboratory Animal Care and Use Committee (LACUC), Wright State University.

Watch this Scientific Journal Video about Rapid Determination of the Thermal Nociceptive Threshold in Diabetic Rats at JoVE.com

Rapid Determination of the Thermal Nociceptive Threshold in Diabetic Rats

Here, we describe a rapid reliable and simple procedure to determine the lowest temperature at which rats or mice show nocifensive behavior, i.e. the thermal nociceptive threshold (TNT). This method applies a slowly increasing thermal stimulus allowing precise and reproducible estimation of TNTs with minimum, if any, stress to the animals.

Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e., increased sensitivity to noxious stimulus, and allodynia i.e., hypersensitivity ...

rapid-determination-thermal-nociceptive-threshold-diabetic

Research

JoVE Journal

Neuroscience

27.4K Views.  Wright State University. Здесь мы рассмотрим быстрый надежный и простой процедурой, чтобы определить самую низкую температуру, при которой крыс или мышей показывают nocifensive поведения, Т.е. Ноцицептивных тепловой порог (ТНТ). Этот метод применяется медленно растущих теп...

Video: Rapid Determination of the Thermal Nociceptive Threshold in Diabetic Rats

Chronic Constriction of the Sciatic Nerve and Pain Hypersensitivity Testing in Rats

Chronic neuropathic pain, resulting from damage to the central or peripheral nervous system, is a prevalent and debilitating condition, affecting 7-18% of the population1,2. Symptoms include spontaneous (tingling, burning, electric-shock like) pain, dysaesthesia, paraesthesia, allodynia (pain resulting from normally non-painful stimuli) and hyperalgesia (an increased response to painful stimuli). The sensory symptoms are co-morbid with behavioural disabilities, such as insomnia and depression. To study chronic neuropathic pain several animal models mimicking peripheral nerve injury have been developed, one of the most widely used is Bennett and Xie's (1988) unilateral sciatic nerve chronic constriction injury (CCI)3 (Figure 1). Here we present a method for performing CCI and testing pain hypersensitivity.
CCI is performed under anaesthesia, with the sciatic nerve on one side exposed by making a skin incision, and cutting through the connective tissue between the gluteus superficialis and biceps femoris muscles. Four chromic gut ligatures are tied loosely around the sciatic nerve at 1 mm intervals, to just occlude but not arrest epineural blood flow. The wound is closed with sutures in the muscle and staples in the skin. The animal is then allowed to recover from surgery for 24 hrs before pain hypersensitivity testing begins.
For behavioural testing, rats are placed into the testing apparatus and are allowed to habituate to the testing procedure. The area tested is the mid-plantar surface of the hindpaw (Figure 2), which falls within the sciatic nerve distribution. Mechanical withdrawal threshold is assessed by mechanically stimulating both injured and uninjured hindpaws using an electronic dynamic plantar von Frey aesthesiometer or manual von Frey hairs4. The mechanical withdrawal threshold is the maximum pressure exerted (in grams) that triggers paw withdrawal. For measurement of thermal withdrawal latency, first described by Hargreaves et al (1988), the hindpaw is exposed to a beam of radiant heat through a transparent glass surface using a plantar analgesia meter5,6. The withdrawal latency to the heat stimulus is recorded as the time for paw withdrawal in both injured and uninjured hindpaws. Following CCI, mechanical withdrawal threshold, as well as thermal withdrawal latency in the injured paw are both significantly reduced, compared to baseline measurements and the uninjured paw (Figure 3). The CCI model of peripheral nerve injury combined with pain hypersensitivity testing provides a model system to investigate the effectiveness of potential therapeutic agents to modify chronic neuropathic pain. In our laboratory, we utilise CCI alongside thermal and mechanical sensitivity of the hindpaws to investigate the role of neuro-immune interactions in the pathogenesis and treatment of neuropathic pain.

Due to the simplicity of surgery and the robust behavioural outcome, chronic constriction of the sciatic nerve is one of the pre-eminent animal models of neuropathic pain. Within 24 hrs following surgery, pain hypersensitivity is established and can be quantitatively measured using a von Frey aesthesiometer (mechanical test) and plantar analgesia meter (thermal test).

Chronic neuropathic pain, resulting from damage to the central or peripheral nervous system, is a prevalent and debilitating condition, affecting 7-18% of ...

Medicine

Determining heat and mechanical pain threshold in inflamed skin of human subjects

In a previous article in the Journal of Visualized Experiments we have demonstrated skin microdialysis techniques for the collection of tissue-specific nociceptive and inflammatory biochemicals in humans. In this article we will show pain-testing paradigms that are often used in tandem with microdialysis procedures. Combining pain tests with microdialysis provides the critical link between behavioral and biochemical data that allows identifying key biochemicals responsible for generating and propagating pain.
Two models of evoking pain in inflamed skin of human study participants are shown. The first model evokes pain with aid of heat stimuli. Heat evoked pain as described here is predominantly mediated by small, non-myelinated peripheral nociceptive nerve fibers (C-fibers). The second model evokes pain via punctuated pressure stimuli. Punctuated pressure evoked pain is predominantly mediated by small, myelinated peripheral nociceptive nerve fibers (A-delta fibers). The two models are mechanistically distinct and independently examine nociceptive processing by the two major peripheral nerve fiber populations involved in pain signaling.
Heat pain is evoked with aid of the TSA II, a commercially available thermo-sensory analyzer (Medoc Advanced Medical Systems, Durham, NC). Stimulus configuration and delivery is handled with aid of specific software. Thermodes vary in size and shape but in principle consist of a metal plate that can be heated or cooled at various rates and for different periods of time. Algorithms assessing heat-evoked pain are manifold. In the experiments shown here, study participants are asked to indicate at what point they start experiencing pain while the thermode in contact with skin is heated at a predetermined rate starting at a temperature that does not evoke pain. The thermode temperature at which a subject starts experiencing pain constitutes the heat pain threshold.
Mechanical pain is evoked with punctuated probes. Such probes are commercially available from several manufacturers (von Frey hairs). However, the accuracy of von Frey hairs has been criticized and many investigators use custom made punctuated pressure probes. In the experiments shown here eight custom-made punctuated probes of different weights are applied in consecutive order, a procedure called up-down algorithm, to identify perceptional deflection points, i.e., a change from feeling no pain to feeling pain or vice versa. The average weight causing a perceptional deflection constitutes the mechanical pain threshold.

Algorithms assessing heat and mechanical pain thresholds in experimentally inflamed skin of human study subjects are shown. The two pain testing paradigms independently examine nociceptive processing by the two major peripheral nerve fiber populations transmitting pain, i.e., non-myelinated C fibers and small myelinated A-delta fibers.

In a previous article in the Journal of Visualized Experiments we have demonstrated skin microdialysis techniques for the collection of tissue-specific ...

Biology

Chronic Post-Ischemia Pain Model for Complex Regional Pain Syndrome Type-I in Rats

Complex regional pain syndrome type-I (CRPS-I) is a neurological disease that causes severe pain among patients and remains an unresolved medical condition. However, the underlying mechanisms of CRPS-I have yet to be revealed. It is known that ischemia/reperfusion is one of the leading factors that causes CRPS-I. By means of prolonged ischemia and reperfusion of the hind limb, the rat chronic post-ischemia pain (CPIP) model has been established to mimic CRPS-I. The CPIP model has become a well-recognized animal model for studying the mechanisms of CRPS-I. This protocol describes the detailed procedures involved in the establishment of the rat model of CPIP, including anesthesia, followed by ischemia/reperfusion of the hind limb. Characteristics of the rat CPIP model are further evaluated by measuring the mechanical and thermal hypersensitivities of the hind limb as well as the nocifensive responses to acute capsaicin injection. The rat CPIP model exhibits several CRPS-I-like manifestations, including hind limb edema and hyperemia in the early stage after establishment, persistent thermal and mechanical hypersensitivities, and increased nocifensive responses to acute capsaicin injection. These characteristics render it a suitable animal model for further investigation of the mechanisms involved in CRPS-I.

Provided here is a protocol that details steps to establish an animal model of chronic post-ischemia pain (CPIP). This is a well-recognized model mimicking human complex regional pain syndrome type-I. Mechanical and thermal hypersensitivities are further evaluated, as well as capsaicin-induced nocifensive behaviors observed in the CPIP rat model.

Complex regional pain syndrome type-I (CRPS-I) is a neurological disease that causes severe pain among patients and remains an unresolved medical ...

Behavior

Assessment of Morphine-induced Hyperalgesia and Analgesic Tolerance in Mice Using Thermal and Mechanical Nociceptive Modalities

Opioid-induced hyperalgesia and tolerance severely impact the clinical efficacy of opiates as pain relievers in animals and humans. The molecular mechanisms underlying both phenomena are not well understood and their elucidation should benefit from the study of animal models and from the design of appropriate experimental protocols. 
We describe here a methodological approach for inducing, recording and quantifying morphine-induced hyperalgesia as well as for evidencing analgesic tolerance, using the tail-immersion and tail pressure tests in wild-type mice. As shown in the video, the protocol is divided into five sequential steps. Handling and habituation phases allow a safe determination of the basal nociceptive response of the animals. Chronic morphine administration induces significant hyperalgesia as shown by an increase in both thermal and mechanical sensitivity, whereas the comparison of analgesia time-courses after acute or repeated morphine treatment clearly indicates the development of tolerance manifested by a decline in analgesic response amplitude. This protocol may be similarly adapted to genetically modified mice in order to evaluate the role of individual genes in the modulation of nociception and morphine analgesia. It also provides a model system to investigate the effectiveness of potential therapeutic agents to improve opiate analgesic efficacy.

We describe a protocol to examine the development of opioid-induced hyperalgesia and tolerance in mice. Based on the measurement of thermal and mechanical nociceptive responses of na&#239;ve and morphine-treated animals, it allows to quantify the increase in pain sensitivity (hyperalgesia) and decrease in analgesia (tolerance) associated with chronic opiate administration.

Opioid-induced hyperalgesia and tolerance severely impact the clinical efficacy of opiates as pain relievers in animals and humans. The molecular ...

Back Mechanical Sensitivity Assessment in the Rat for Mechanistic Investigation of Chronic Back Pain

Low back pain is the leading cause of disability worldwide, with dramatic personal, economic, and social consequences. To develop novel therapeutics, animal models are needed to examine the mechanisms and effectiveness of novel therapies from a translational perspective. Several rodent models of back pain are used in current investigations. Surprisingly, however, no standardized behavioral test was validated to assess mechanical sensitivity in back pain models. This is critical to confirm that animals with presumed back pain present local hypersensitivity to nociceptive stimuli, and to monitor sensitivity during interventions designed to relieve back pain. The objective of this study is to lay down a simple and accessible test to assess mechanical sensitivity in the back of rats. A test cage was fabricated specifically for this method; length x width x height: 50 x 20 x 7 cm, having a stainless-steel mesh on the top. This test cage allows the application of mechanical stimuli to the back. To perform the test, the back of the animal is shaved in the region of interest, and the test area is marked to repeat the test on different days, as needed. The mechanical threshold is determined with Von Frey filaments applied to the paraspinal muscles, utilizing the up-down method described previously. The positive responses include (1) muscle twitching, (2) arching (back extension), (3) rotation of the neck (4) scratching or licking the back, and (5) escaping. This behavioral test (Back Mechanical Sensitivity (BMS) test) is useful for mechanistic research with rodent models of back pain for the development of therapeutic interventions for the prevention and management of back pain.

To develop novel therapeutic interventions for the prevention and management of back pain, animal models are required to examine the mechanisms and effectiveness of these therapies from a translational perspective. The present protocol describes the BMS test, a standardized method to assess back mechanical sensitivity in the rat.

Low back pain is the leading cause of disability worldwide, with dramatic personal, economic, and social consequences. To develop novel therapeutics, ...

Assessment of Nerve Injury-Induced Mechanical Hypersensitivity in Rats Using an Orofacial Operant Pain Assay

Pain has sensory and affective components. Unlike traditional, reflex-based pain assays, operant pain assays can produce more clinically relevant results by addressing the cognitive and motivational aspects of pain in rodents. This paper presents a protocol for assessing mechanical hypersensitivity following chronic constriction injury of the infraorbital nerves (CCI-ION) in rats using an orofacial operant pain system. Before CCI-ION surgery, rats were trained in an orofacial pain assessment device (OPAD) to drink sweetened condensed milk while making facial contact with the metal spiked bars and lick-tube.
In this assay, rats can choose between receiving milk as a positive reinforcer or escaping an aversive mechanical stimulus that is produced by a vertical row of small pyramid-shaped spikes on each side of the reward access hole. Following 2 weeks of training in the OPAD and before the CCI-ION surgery, baseline mechanical sensitivity data were recorded for 5 days for each rat during a 10 min testing session. During a session, the operant system automatically records the number of reward bottle activations (licks) and facial contacts, contact duration, and latency to the first lick, among other measures.
Following baseline measurements, rats underwent either CCI-ION or sham surgery. In this protocol, mechanical hypersensitivity was quantified by measuring the number of licks, latency to the first lick, the number of contacts, and the ratio of licks to facial contacts (L/F). The data showed that CCI-ION resulted in a significant decrease in the number of licks and the L/F ratio and an increase in the latency to the first lick, indicating mechanical hypersensitivity. These data support the use of operant-based pain assays to assess mechanical pain sensitivity in preclinical pain research.

This protocol describes the assessment of mechanical hypersensitivity in a rat model of neuropathic orofacial pain using an operant-based orofacial pain assessment device.

Pain has sensory and affective components. Unlike traditional, reflex-based pain assays, operant pain assays can produce more clinically relevant results ...

An Ultrasonic Tool for Nerve Conduction Block in Diabetic Rat Models

Nerve conduction block with a high intensity-focused ultrasound (HIFU) transducer has been performed in normal and diabetic animal models recently. HIFU can reversibly block the conduction of peripheral nerves without damaging the nerves while using an appropriate ultrasonic parameter. Temporary and partial block of the action potentials of nerves shows that HIFU has the potential to be a useful clinical treatment for pain relief. This work demonstrates the procedures for suppressing the action potentials of neuropathic nerves in diabetic rats in vivo using an HIFU transducer. The first step is to generate adult male diabetic neuropathic rats by streptozotocin (STZ) injection. The second step is to evaluate the peripheral diabetic neuropathy in STZ-induced diabetic rats by an electronic von Frey probe and a hot plate. The final step is to record in vivo extracellular action potentials of the nerve exposed to HIFU sonication. The method showed here may benefit the study of ultrasound analgesic applications.

This work presents the methodology of applying high intensity-focused ultrasound to block the action potentials of diabetic neuropathic nerves.

Nerve conduction block with a high intensity-focused ultrasound (HIFU) transducer has been performed in normal and diabetic animal models recently. HIFU ...

Bioengineering

Modified Spared Nerve Injury Surgery Model of Neuropathic Pain in Mice

Spared nerve injury (SNI) is an animal model that mimics the cardinal symptoms of peripheral nerve injury for studying the molecular and cellular mechanism of neuropathic pain in mice and rats. Currently, there are two types of SNI model, one to cut and ligate the common peroneal and the tibial nerves with intact sural nerve, which is defined as SNIs in this study, and another to cut and ligate the common peroneal and the sural nerves with intact tibial nerve, which is defined as SNIt in this study. Because the sural nerve is purely sensory whereas the tibial nerve contains both motor and sensory fibers, the SNIt model has much less motor deficit than the SNIs model. In the traditional SNIt mouse model, the common peroneal and the sural nerves are cut and ligated separately. Here a modified SNIt surgery method is described to damage both common peroneal and sural nerves with only one ligation and one cut with a shorter procedure time, which is easier to perform and reduces the potential risk of stretching the sciatic or tibial nerves, and produces similar mechanical hypersensitivity as the traditional SNIt model.

The modified surgery is a simplified method for mouse or rat spared nerve injury model that requires only one ligation and one cut to injure both common peroneal and sural nerves.

Spared nerve injury (SNI) is an animal model that mimics the cardinal symptoms of peripheral nerve injury for studying the molecular and cellular ...

Assessing Tuina Therapy&#039;s Pain Relief in Chronic Constriction Rats

Characteristics of Pain Changes in Rats with Nerve Injury Within 24 hours After One-Time Tuina Intervention

Tuina, as an external treatment method of traditional Chinese medicine, has been proven to have an analgesic effect on peripheral neuropathic pain (pNP) in clinical and basic research. However, the optimal time point for the analgesic effect of tuina may vary according to different injury sensations, affecting the exploration of the initiation mechanism of tuina analgesia. 
The research used minor chronic constriction injury (minor CCI) model rats to simulate pNP and used the intelligent tuina manipulation simulator to simulate the three methods (point-pressing, plucking, and kneading) and three acupoints (Yinmen BL37, Chengshan BL57, and Yanglingquan GB34) for performing tuina therapy. The study evaluated the changes in pain within 24 h and the optimal time point for the efficacy of tuina analgesia in rats with minor CCI models by testing cold sensitivity threshold (CST), mechanical withdrawal threshold (MWT), and thermal withdrawal latency (TWL). Furthermore, the study evaluated IL-10 and TNF-&#945; expression changes through Elisa detection. The results show that tuina has both immediate and sustained analgesic effects. For the three different injury sensitivity thresholds of CST, MWT, TWL, and two cytokines of IL-10 and TNF-&#945;, the analgesic efficacy of tuina within 24 h after intervention is significantly different at different time points.

Author Spotlight: Exploring the Initiation Mechanism of &#039;Three-Method and Three-Acupoint&#039; Tuina Analgesia in Rats with Nerve Injury

We present a protocol using the tuina manipulation simulator to perform the "Three-Manipulation and Three-Acupoint" tuina therapy for minor chronic constriction injury rats and evaluate the effective analgesic time points of tuina within 24 h by testing pain changes through behavioral analysis and changes in inflammatory factor expression using enzyme-linked immunosorbent assay.

Tuina, as an external treatment method of traditional Chinese medicine, has been proven to have an analgesic effect on peripheral neuropathic pain (pNP) ...

Partial Sciatic Nerve Ligation: A Mouse Model of Chronic Neuropathic Pain to Study the Antinociceptive Effect of Novel Therapies

Management of chronic pain remains challenging to this day, and current treatments are associated with adverse effects, including tolerance and addiction. Chronic neuropathic pain results from lesions or diseases in the somatosensory system. To investigate potential therapies with reduced side effects, animal pain models are the gold standard in preclinical studies. Therefore, well-characterized and well-described models are crucial for the development and validation of innovative therapies. 
Partial ligation of the sciatic nerve (pSNL) is a procedure that induces chronic neuropathic pain in mice, characterized by mechanical and thermal hypersensitivity, ongoing pain, and changes in limb temperature, making this model a great fit to study neuropathic pain preclinically. pSNL is an advantageous model to study neuropathic pain as it reproduces many symptoms observed in humans with neuropathic pain. Furthermore, the surgical procedure is relatively fast and straightforward to perform. Unilateral pSNL of one limb allows for comparison between the ipsilateral and contralateral paws, as well as evaluation of central sensitization. 
To induce chronic neuropathic hypersensitivity, a 9-0 non-absorbable nylon thread is used to ligate the dorsal third of the sciatic nerve. This article describes the surgical procedure and characterizes the development of chronic neuropathic pain through multiple commonly used behavioral tests. As a plethora of innovative therapies are now being investigated to treat chronic pain, this article provides crucial concepts for standardization and an accurate description of surgeries required to induce neuropathic pain.

Partial sciatic nerve ligation induces long-lasting chronic neuropathic pain, characterized by exaggerated responses to thermal and mechanical stimuli. This mouse model of neuropathic pain is commonly used to study innovative therapies for pain management. This article describes in detail the surgical procedure to improve standardization and reproducibility.

Management of chronic pain remains challenging to this day, and current treatments are associated with adverse effects, including tolerance and addiction. ...